Construction and Characterization of Retroviral Vectors Expressing Biologically Active Human Interleukin-12

Abstract

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hlL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neo^R) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10⁶ cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10⁶ cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-γ (IFN-γ) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer. Therapeutic tumor vaccines using autologous tumor cells genetically engineered to produce cytokines have been developed in murine models and are now being tested in clinical trials. Emerging experimental evidence suggests that interleukin-12 (IL-12) can enhance the development of an effective immune response against tumors as well as certain infectious agents. Thus, IL-12 is an appropriate cytokine to test in therapeutic clinical studies using local delivery via gene transfer either directly into tumor cells or, alternatively, in fibroblast carriers. Retroviral vectors have been constructed to express coordinately both chains, p35 and p40, of human IL-12 (hIL-12) from a polycistronic message using the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV). Infection of both mouse and human tumor cell lines resulted in production of high levels of bioactive IL-12. Whereas professional antigen-presenting cells (APC) so far have been the only cells able to normally excrete IL-12, retrovirally infected tumor cell lines and fibroblasts secrete substantially higher levels of bioactive IL-12. These IL-12 retroviral vectors are suitable for use in clinical trials for gene therapy of cancer.